The focus of this proposal is to investigate the feasibility for therapeutic intervention in Huntington's Disease (HD) through modulation of the rate of degradation of mutant Huntingtin (Htt) protein. In earlier studies, we have demonstrated that modification of specific amino terminal residues of the Htt protein dramatically influences the rate of degradation of Htt via both the proteosomal and lysosomal pathways. Phosphorylation of serine (S) 13, a reaction which can be catalyzed by the kinase IKKb, in turn leads to phosphorylation of S16 and acetylation on lysine (K) 9. Phosphorylation at S13 is much more efficient for wild type Htt than for the expanded mutant form. We hypothesize that clearance of phosphorylated mutant Htt is impaired upon aging, when clearance mechanisms are no longer as efficient, causing its accumulation and contributing to the onset of HD symptoms and pathogenesis. We now propose to carry out an integrated series of studies which seek to explore this hypothesis in depth. We hope to provide a basis for therapeutic intervention for HD based on detailed understanding of the mechanisms which regulate Htt degradation and distinguish mutant Htt from normal Htt with respect to degradation. Specifically, we will investigate the following specific aims: Specific Aim 1: Molecular and temporal relationship of Htt phosphorylation and acetylation in the progression of HD in vivo. We have showed that phosphorylation of Htt S13 activates subsequent S16 phosphorylation and K9 acetylation and regulates its degradation, aggregation and toxicity. We will now determine whether the temporal sequence of these modifications of Htt in mammalian brain is significantly correlated with the temporal sequence of clinical pathology in HD. We will therefore quantitate the levels, distribution, size and solubility of phosphorylated and acetylated Htt in human brain material isolated at autopsy from individuals at different stages of HD progression. These studies will be complemented by studies with a similar design carried out on mouse models for HD in which a temporal sequence of neuropathology is well established. Specific Aim 2: Test the hypothesis that IKK phosphorylates Htt in vivo. IKK can directly phosphorylate Htt, activate its acetylation and clearance and regulate its solubility. We now propose to determine whether IKK modulates these processes in mouse brain in vivo. Specific Aim 3: Investigate phosphorylation of Htt by the kinases Akt and Cdk5 and determine whether phosphorylation of Htt by IKK, Akt and/or Cdk5 are interdependent. Previous studies in cell based systems have shown that phosphorylation of Htt by Akt and Cdk5 reduces Htt-mediated toxicity and mutant Htt aggregation which may suggest that these phosphorylation events facilitate Htt clearance We will now investigate the extent to which Akt, Cdk5 and IKK-mediated phosphorylation and degradation are interdependent in both cell culture systems and in vivo via brain tissue analysis.